M5 muscarinic acetylcholine receptors (mAChRs) have emerged as a potential target for the treatment of drug abuse, based on brain region localization, involvement in the regulation of central dopaminergic pathways, and behavioral data from M5-knockout mice. However, the exact physiological role of this receptor and its potential for pharmacotherapeutic development are ambiguous due to the lack of selective ligands. The purpose of the current proposal is to develop novel M5 receptor antagonists. Based on a reported nonselective muscarinic antagonist, we generated over 70 structurally-related analogs using a progressive step-by-step structural modification strategy from which several new leads with increased selectivity and potency for M5 mAChR subtypes have been identified. Further, we have constructed a homology model of M5 mAChR based on the newly available crystal structure of the b1 adrenergic receptor. In this revision, we propose to build and validate homology models for the other 4 mAChR subtypes using reported compounds. These in silico models will be used for virtual library screening. A structurally diversified virtual screening library, which consists of 9 different structural scaffolds, was designed and anticipate that highly selective and potent analogs as virtual hits for M5 mAChRs will emerge. The virtual hits will be pre-evaluated in in silico ADMET screening programs to focus the synthetic efforts on druggable analogs. Druggable virtual hits will be evaluated in receptor binding assays using CHO cells expressing hM1-hM5. Analogs selective for M5 will be evaluated in functional assays using native M5 receptors and lead analogs will be evaluated also in off-target assays, for in vitro cardiotoxicity (hERG assays) and neurotoxicity (dopamine striatal content). We anticipate the discovery of promising M5 antagonists, which will be useful pharmacological tools and have potential as novel therapeutic agents for the treatment of drug abuse.